Method for engineering strand-specific nicking endonucleases from restriction endonucleases
US7943303B2 · kind B2 · utility
Assignee
Inventors
Key dates
| Filing date | Dec 15, 2004 |
| Grant date | May 17, 2011 |
| Priority date | — |
| Expiry date | Dec 15, 2024 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12Q1/44
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
Methods are provided for engineering novel strand-specific nicking endonucleases by means of an in vivo enrichment of a plasmid library containing a randomly mutagenized restriction endonuclease gene. The plasmids contain adjacent to the gene a cleavable or nickable sequence for cleaving or nicking by the endonuclease product of the gene and a second recognition site for a second endonuclease. The plasmid library is used to transform unmodified host cells. Plasmids from the cultured transformed cells may be analyzed by an in vitro assay for nicking and the nicked plasmids pooled and used to transform host cells. The product is then pooled and the single-stranded specificity of the endonuclease is then determined. The product is either cloned after amplification or identified by use of a selectable marker.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.